It has long been a problem with cultivating cells that they require iron as a necessary component in the medium. To present the iron molecules to the cell membrane, it has previously been necessary to add transferrin, usually through addition of serum, to the growth medium. However, such an addition of serum will also produce several disadvantages.
It has been found that aurintricarboxylic acid will present iron to the cell membrane equally well, thus avoiding the addition of serum to the growth medium.
However, to bring the iron into a form which may avoid that the element precipitating from the solution, it is necessary to add chelating agents to the medium. This, taken together with the action of aurintricarboxylic acid, makes it preferable to add EDTA and citric acid/citrate to the medium (or the additive according to the invention, whichever the case may be). Thus, the citric acid is not added to adjust the pH, but is added as a chelator (though it may incidentally also have pH-adjusting properties as well). However, EDTA is just an example of a chelating agent to make a stable iron chelate, and any biocompatible chelating agent (and several are known in the art of cultivating cells) may be used. As mentioned above, the main consideration is that aurintricarboxylic acid presents the iron in solution to the cell membrane.
The present invention concerns a serum-free medium which may be used in culturing cells which require iron in the growth medium. The invention may be used to test the quality of other growth media in addition to that it is possible to culture cells in media which are free from serum.
There has in industry and research long been a large need of serum-free media and growth cultures since the supply of serum to such media gives the solutions, in addition to the compounds which are necessary for growth, also compounds that influence most expriments negatively where accracy of the results are of great importance. This is valid within discipline such as cellular immunology, biotechnology, "in vitro" fertilization, organ transplants, cancer research and by storing and transfusion of blood.
Those media and physiological solutions which today are used in research, industry and by clinical work, are today based on up to 100 year old recipies, and represent thus a "stand-still" in this field of biological research. Media used until today for cell cultures comprise most often a so-called "commercial base medium" to which there is added approximately 10% heat inactivated serum. This very old method comprises the following disadvantages:
1) The heat treatment which is necessary to prevent the lytic effect of the serum on cells, has a denaturing effect on important serum components.
2) Different serum batches have different properties on account of their origin, something which adds unwanted variations to the behaviour of the cell cultures "in vitro".
3) Serum contains several unknown factors and compounds with unknown and uncontrolled effect on cells.
4) Serum is an unphysiological fluid for most cells since the cells are adapted to the so-called tissue-fluid in the body, whereby this fluid has a different content of different compounds compared to serum.
5) Antibodies in serum may bind to cells and interfere with experiments.
6) Serum prevents the study of the cell's synthesis of serum factors "in vitro" since the "background level" in serum of such factors is relatively high.